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Mycobacterium avium complex (MAC) disease has been increasing worldwide not only in immunocompromised but also in immunocompetent humans. However, the relationship between mycobacterial strain virulence and disease progression in immunocompetent humans is unclear. In this study, we isolated 6 strains from patients with pulmonary MAC disease. To explore the virulence, we examined the growth in human THP-1 macrophages and pathogenicity in C57BL/6 mice. We found that one strain, designated 198, which was isolated from a patient showing the most progressive disease, persisted in THP-1 cells. In addition, strain 198 grew to a high bacterial load with strong inflammation in mouse lungs and spleens 16 weeks after infection. To our knowledge, strain 198 is the first isolated MAC strain that exhibits hypervirulence consistently for the human patient, human macrophages in vitro, and even for immunocompetent mice. Other strains showed limited survival and weak virulence both in macrophages and in mice, uncorrelated to disease progression in human patients. We demonstrated that there is a hypervirulent clinical MAC strain whose experimental virulence corresponds to the serious disease progression in the patients. The existence of such strain suggests the involvement of bacterial virulence in the pathogenesis of pulmonary MAC disease in immunocompetent status.

Factors that influence colony morphology are of crucial importance for drug development as well as for understanding the virulence of Mycobacterium avium complex (MAC) strains. The MAC 101 strain used in the present study grows as smooth transparent (SmT) colonies that tend to become opaque and pigmented when incubated for long periods of time. However, when MAC was passaged in animals, two types of colonies were recovered. The new rough transparent (RgT) colony morphology appeared more flat and transparent, having a central spot, irregular edges at times, and a dry, granular appearance like that of the rough mutants. In animal studies, the RgT bacilli multiplied at a much faster rate than that of the SmT bacilli, causing 60-80% mortality compared with the 10% mortality observed in mice infected with SmT. In vitro studies indicated that the SmT MAC did not grow and multiply as well in resident peritoneal macrophages as the RgT MAC did. The two morphotypes did not differ in their growth ratesin vitro but the RgT MAC failed to reduce dimethylthiazol-diphenyltetrazolium bromide (MTT), alamar blue and neutral red, suggesting that there might be significant changes in the cell wall or elsewhere causing changes in cellular permeability. These two morphotypes could serve as models for studying the biochemical markers or the identification of factors responsible for the virulence of the MAC., (Copyright 1998 Academic Press.)

The Mycobacterium avium complex (MAC) includes the closely related species M. avium, M. intracellulare and M. paratuberculosis. The insertion elements IS900, IS901, IS1245 and IS1311 were used as DNA probes to characterize by restriction fragment polymorphisms (RFLPs) eight reference strains, three animal isolates of M. paratuberculosis from outside New Zealand and 61 selected New Zealand MAC isolates from cattle, deer, pigs, sheep and humans. IS900 was found only in strains of M. paratuberculosis. All MAC strains contained IS1311 and the RFLPs associated with this insertion element divided M. paratuberculosis strains into the same groups as IS900 RFLPs. Except for M. paratuberculosis, all MAC strains contained IS1245 and the majority of those from lesions in cattle, deer and pigs also contained IS901. All animal strains containing IS901 had the same RFLPs with IS901, IS1245 and IS1311. In three cases, these apparently identical strains could be differentiated by restriction fragment analysis with BstEII. IS901 was not present in four human isolates or in isolates from deer without lesions. These results indicate that a very closely related group of strains causes the majority of non-paratuberculosis MAC lesions in animals in New Zealand.

Mycobacterium avium complex (MAC) is an intracellular pathogen which causes disseminated bacterial infection in immunocompromised individuals. This organism predominantly infects macrophages. Attachment of MAC to macrophages is the first step prior to invasion. We have previously shown that a 70 kDa protein of M. avium (Ma) is one of nine monocyte-binding proteins. In the present study, we have purified this protein from sonic extracts of Ma and studied some of its properties. The N-terminal sequence of this protein was identified and found to exhibit a strong homology to the 70 kDa heat shock protein (hsp) of M. leprae (Ml) and M. tuberculosis (Mtb). This protein was found to be present on the surface of the organism and was able to inhibit the attachment of intact Ma to human monocyte derived macrophages (MDM) up to 49% in an in vitro attachment assay using intact fluorescein isothiocyanate (FITC)-labelled Ma. Bovine serum albumin (BSA) and recombinant 70 kDa hsp from Mtb, which were used as controls, inhibited this attachment by 9.8 and 18%, respectively. These results suggest that the 70 kDa protein may have a role in the attachment of intact Ma to MDM. When tested in lymphocyte activation assays, this protein did not appear to significantly stimulate proliferation. However, it was found to stimulate the production of tumor necrosis factor (TNF)-alpha by MDM. This protein may be one of several Ma antigens that trigger host immune response by binding to MDM and stimulating the production of inflammatory cytokines such as TNF-alpha by these cells.

Mycobacterium avium complex (MAC) strains are known to exhibit variation in colony morphology. In addition to the smooth transparent (ST), smooth opaque (SO) and rough opaque (RO), which are the most common morphological forms, intermediate (IM) and pin point (PP) forms were also occasionally observed. In order to understand the pathobiological significance of these different colony forms, we investigated their virulence in beige mice, ability to bind to plastic and epithelial cells, differences in the lipids, and modulation of macrophage functions by the bacillary extracts. ST variants, the most common form seen in AIDS patients, were more virulent with increased multiplication in lungs, livers and spleens of beige mice and showed increased adherence to plastic and epithelial cells. SO, RO, PP colonial forms did not show increase in growth in any of the organs over a period of 4 weeks. IM colonial variants showed increased growth in lungs and spleens but not in livers. Thin layer chromatographic (TLC) analysis of lipid extracts showed one specific component in the high polar lipids of the SO variant, while ST variant did not show any specific component in any of the three families of lipids (high, intermediate and low polarity). The RO variant either expressed low levels or lost many of the components of lipids of high and intermediate polarity, however produced increased levels of lipids of low polarity. One of the components of low polar lipids was specific for RO variant and was produced in large quantity. The isogenic variants differed in the total lipid and sugar contents and also differed in their ability to modulate macrophage functions.

Mycobacterium avium complex (MAC) strains from AIDS and non-AIDS patients and from the environment were studied for their colony morphology and virulence in beige mice. The majority of the MAC isolates from AIDS patients, in contrast to those from non-AIDS patients and the environment, showed increased virulence. Similarly, the majority of the MAC isolates from AIDS patients formed smooth transparent (ST) colonies, whereas most of the non-AIDS isolates formed smooth opaque (SO) or intermediate (IM) type of colonies. MAC isolates from the same AIDS patient obtained at different times were found to be heterogenic with respect to serotype, RFLP and glycolipid patterns, suggesting that these patients might be infected with more than one strain of MAC.

The virulence of 24 strains of Mycobacterium avium complex (MAC) isolated from patients with acquired immune deficiency syndrome (AIDS) was assessed using the beige mouse model. Most changes in colony forming unit (cfu) counts in spleen and lungs, and spleen weights occurred between days 1 and 14, with comparatively smaller changes 14-28 days postinfection. The virulence was assessed by a score formulated from the four most useful parameters: mortality, spleen cfu, lung cfu and spleen weights at 28 days. The scores of the 24 strains showed a normal distribution; four strains falling above one standard deviation from the mean were classified as high virulent, those four falling below one standard deviation as low virulent, and the remaining 16 as of intermediate virulence. Virulence was associated with the total number of plasmids and the occurrence of large plasmids (greater than 100 MDa) in the MAC strains. There was an inverse correlation between virulence and the organism's capacity to trigger the release of oxygen metabolites from peritoneal macrophages. Macrophages from mice infected with the MAC strains of different degrees of virulence released superoxide anion (O2-) with a peak at two weeks, the peak levels bearing an inverse correlation to virulence. No association was seen between virulence and source of specimens, biochemical characteristics, drug susceptibility, serotypes or phage types.